Discussion
The M. arvalis populations are characterized by large-scale genetic differentiation of Tshr , reflecting local adaptation to annual temperature-photoperiod patterns, rather than latitude perse. Variation in Tshr sequence indicates that the M. arvalis population can be subdivided into Eastern and Western European clusters, indicating that they may belong to distinct genetic lineages (Figs. 3, S3). This phylogeographical structure is consistent with that found for mitochondrial cytochrome b gene sequences, and microsatellite loci (representing nuclear DNA)(Haynes, Jaarola, & Searle, 2003; Martínková et al., 2013; Stojak et al., 2016; Stojak, Mcdevitt, Herman, Searle, & Wójcik, 2015), and justifies the analysis of Western and Eastern European populations separately. The Western versus Eastern divide could well be due to re-invasion of Northern Europe from separate glacial refugia, and therefore separate evolutionary events(Hewitt, 1999).
For insights into the geographical variation in Tshr , the association of SNP frequencies with local climatic conditions was examined. Here we showed that genetic variation in the vole Tshris better explained by local photoperiod-temperature patterns than by latitude only. This may be caused by the temperature dependence of vegetation growth. In house mice, genes (but not the Tshr ) that show signals of selection are also associated with local average annual ambient temperature, and are linked with clinal variation in phenotypic aspects, such as body mass and metabolism(Ferris et al., 2021; Phifer-Rixey et al., 2018). Interestingly, SNPs found in thyroid hormone receptors, which are involved in regulation of seasonal reproduction in the hypothalamus(Yoshimura et al., 2003), significantly correlated with variation in average annual temperature(Ferris et al., 2021). This suggests that genomic evolution of seasonal adaptation in house mice and voles involves unique responses to genetic selection. Annual temperature patterns not only depend on latitude, but also on longitude, altitude, and other regional climatic variables like the Gulf Stream warming European Atlantic coastal regions. Critical photoperiods in pitcher-plant mosquitoes strongly correlated with altitude-corrected latitude (r = 0.96), however, this measure does not integrate local temperature patterns(W. E. Bradshaw et al., 2006; William E. Bradshaw, 1976; William E. Bradshaw & Lounibos, 1977). Deriving the regional photoperiod-temperature ellipsoids may be better to account for such regional climatic differences than latitude or altitude-corrected latitude only. We post-hoc tested photoperiods at other temperature thresholds, however this did not improve the results. Moreover, 6.6°C is not an unreasonable temperature since grass growth is initiated at 5-10°C air temperature(Cooper, 1964; Peacock, 1975, 1976).
In addition, several SNPs correlated well with longitude and altitude (Fig. 4G,J). Altitudinal gradients in seasonal timing of breeding have been observed in deer mice (Peromyscus maniculatus borealis ), with shorter breeding seasons at high elevations(Millar & Innes, 1985). The pCPP at which a temperature threshold for grass growth initiation is reached can be deduced from local photoperiod-temperature patterns, and is here confirmed to be a strong determinant for distributional variation in Tshr SNP frequency in Western European common vole populations (Fig. 4M). Pairwise multilocus FST analysis revealed that populations which differ in pCPP, also show greater genetic distance in Tshr haplotypes (Fig. 3). These findings indicate that seasonality is likely to be a selective force forTshr evolution in common voles, and imply that Tshr is an important gene for genetic adaptation of the photoperiodic response systems.
The observed genetic Tshr variation is unlikely to be caused by isolation only, with the possible exception of the Orkney island populations, which are geographically isolated from each other and from mainland populations by the sea. Therefore isolation and genetic drift may be a more important evolutionary force than natural selection in the Orkney populations. Interestingly, the same SNPs appear to be related to pCPP when the Orkney Island populations are excluded from the analysis. This indicates that the results in Western Europe are not dominated by the Orkney population’s data, and that the observed distribution ofTshr variation may be a sign of adaptive evolution likely operating in response to photoperiod.
In Eastern European populations, none of the tested environmental proxies are good predictors for Tshr SNP frequencies (Fig. 5). These results indicate that the Tshr in the Eastern European lineage is not linked to seasonal adaptation as observed in the Western European lineage. Oceanic climates (Western Europe) are known for their small annual temperature amplitudes, while continental climates (Eastern Europe) are known for their large annual temperature amplitudes. These climatic differences may have led to divergent evolutionary adaptation of TSHR function, which may provide an explanation for the observed longitudinal separation in genetic Tshr differentiation. Another hypothesis is that photoperiodic genes other than the Tshr are under selection for seasonal adaptation in Eastern European vole populations.
SNPs associated with local pCPP were all synonymous or intronic mutations. This suggests that these sites may be involved in regulatory rather than structural variation. Five intronic SNPs were strongly associated with pCPP in Western Europe (Fig. 4), of which two (i.e. SNP-144 and -158) were strongly associated with altitude in Eastern Europe (Fig. 5). Putative regulatory protein binding sites were predicted for the intronic region, and revealed that intronic SNP-128, which strongly correlates to pCPP (Fig. 4O), is located in a potential SP1 (specificity protein 1) binding site(Höller, Westin, Jiricny, & Schaffner, 1988; Ji, Casinghino, McCarthy, & Centrella, 1997). Interestingly, SNPs closely located to this enhancer region, such as SNP-158, are related to different environmental proxies in Eastern and Western Europe (Fig. 4, 5, S2). It is tempting to speculate that variation in and around this SP1 binding site sequence may influenceTshr transcription. Furthermore, there is strong evidence that synonymous SNPs are not necessarily neutral as they can alter mRNA expression, splicing, and structure, therefore having downstream effects on protein expression(Chamary, Parmley, & Hurst, 2006; Hunt, Sauna, Ambudkar, Gottesman, & Kimchi-Sarfaty, 2009). Synonymous polymorphisms require different transfer RNAs (tRNA) to recruit the same amino acids and may cause codon-bias. Synonymous tRNA vary strongly in frequency between species and tissues (i.e. codon bias)(Dittmar, Goodenbour, & Pan, 2006; Goodenbour & Pan, 2006). It is therefore possible that the observed synonymous mutations in the TSHR may alter translation efficiency within a species and tissue by changing the elongation rate(Quax, Claassens, Soll, & Oost, 2015). Reduced elongation rate may therefore result in lower protein abundance. Hence synonymous SNPs in the Tshr gene could result in altered receptor abundance, changed sensitivity to TSH and modified photoperiodic response. It is therefore conceivable that synonymous SNPs in the Tshr gene are subject to natural selection, and reflect local geographical adaptation. TSHR plays a pivotal role in photoperiodic response in the pars tuberalis, but also in thyroid hormone metabolism in the thyroid gland. Tissue-specific functions of TSHR may benefit from genetic adaptation in photoperiodism through synonymous SNPs, since tissue-specific tRNA expression, which has been demonstrated in human and mouse tissues(Dittmar et al., 2006; Pinkard, McFarland, Sweet, & Coller, 2020), may perhaps lead to altered TSHR function in the pars tuberalis, but not in the thyroid gland.
Photoperiodic regulation of the reproductive system in deer mice has been shown to vary with latitude, with weaker photoperiodic responses in animals originating from lower latitudes(Dark et al., 1983). Moreover, photoperiodic sensitivity in pitcher-plant mosquitoes correlated with global warming, indicating the importance of season-length driving evolution (genetic change) of photoperiodism during recent rapid climate change(W. E. Bradshaw & Holzapfel, 2008; William E. Bradshaw & Holzapfel, 2001a, 2006; William E. Bradshaw, Zani, & Holzapfel, 2004). Our findings confirm that the Tshr gene is under selection, which has previously been reported in chicken domestication in relation to photoperiodic responsiveness(Karlsson et al., 2016; Rubin et al., 2010). Future studies should determine whether the SNPs identified as seasonal timing dependent genetic variation in the vole Tshr can indeed alter, genetically based, photoperiodic responses. Such an approach will confirm whether habitat-specific photoperiodic responses are indeed regulated by means of functional TSHR adaptation. In vole populations with later onsets of reproduction and shorter breeding seasons(Tkadlec, 2000), our results predict lower concentrations in the tanycytes ofTshr or lower TSH-binding affinities of Tshr haplotypes.
Optimal timing of reproduction, enhancing energetically demanding pregnancy, and parental care, is necessary to maximize fitness in temperate and northern seasonal environments. Tshr is an essential gene in the pathway programming seasonal reproduction in mammals. Herein, we show how onset of the favorable season over a wide geographical range of the common vole, Microtus arvalis , explains much of the genetic variation in the TSH binding site, hinge region, and transmembrane domain of TSHR in Western but not Eastern Europe. Yet, vole populations thrive in both regions. We therefore conclude that different genetic mechanisms have been important in enabling vole populations to exploit geographically distinct regions. Such distinctions of how the genetic underpinnings of seasonal timing have evolved over climatic gradients in nature will be important in predicting how animals will adapt to new seasonal environments during ongoing rapid climate change.